Techniques for Efficient Finishing and Physical Linkage of Gene-Enriched Shotgun Sequences

这一概念验证项目的总体目标是开发玉米基因组资源，将其他正在进行的项目(DBI-0221536[PI：舒伯特]和DBI-0211851[PI：Messing])提供的序列连接在一起。玉米基因组的长度约为34亿个碱基对(MB)，但基因仅覆盖约150-450MB或基因组的20%。初步结果表明，剩下的80%的基因组由重复的DNA组成，其中大部分高度甲基化。这些基因被认为以小岛的形式分布在重复DNA的海洋中。分离和测序基因的方法目前正在评估中，因为目前解决整个基因组问题既不可行，也不划算。该项目的第一部分将使用低甲基化部分限制(HMPR)文库来连接基因内的小甲基化区域，这些区域在基因浓缩方法中丢失，这些方法基于甲基化来过滤重复的DNA。HMPR序列可以通过弥合这些差距来组装连续的基因序列。这个项目的第二部分是产生和测序跨越链接器文库(MSLL)的甲基化，这将在甲基化重复DNA的海洋中提供一座桥梁，从一个基因岛到另一个基因岛。将从两端对HMPR和MSLL文库克隆进行测序，并将这些序列与现有的玉米基因组序列组装在一起。最终结果将有助于玉米基因组图上丰富基因序列支架的开发。所有序列信息将立即发布到GenBank。将在http://www.genome.arizona.edu上创建一个基于网络的展示，以提供对项目结果的访问。

The overall goal of this proof-of-concept project is to develop genomic resources for maize that will link together sequences available from other ongoing projects (DBI-0221536 [PI: Schubert] and DBI-0211851 [PI: Messing]). The maize genome is approximately 3,400 million base pairs (MB) in length, yet the genes only cover about 150-450 MB or 20% of the genome. Preliminary results indicate the remaining 80% of the genome comprises repetitive DNAs, much of which is highly methylated. The genes are thought to be distributed as small islands throughout the sea of repetitive DNA. Methods to isolate and sequence the genes are currently being evaluated since it is not feasible or cost-effective to tackle the whole genome at this time. The first part of this project will use HypoMethylated Partial Restriction (HMPR) libraries to bridge small methylated regions within genes that are lost in gene enrichment methods that filter out repetitive DNA based on methylation. The HMPR sequences could allow contiguous gene sequences to be assembled by bridging these gaps. The second part of this project is to generate and sequence Methylation Spanning Linker Libraries (MSLLs) that will provide a bridge over the sea of methylated repetitive DNA from one gene island to the next. The HMPR and MSLL library clones will be sequenced from both ends and the sequences assembled with the available maize genomic sequence. The end results should contribute to the development of a scaffold of gene-rich sequences positioned across the maize genome map.All sequence information will be released immediately to GenBank. A web-based display will be created at http://www.genome.arizona.edu to provide access to the project results.